Method for selectively detecting dopamine based on magnetic resonance nuclear spin singlet state

ABSTRACT

The present invention discloses a method for selectively detecting dopamine based on magnetic resonance nuclear spin singlet state. The method uses the nuclear spin singlet of three hydrogen atoms on the dopamine benzene ring to achieve selective detection of dopamine signals in a complicated system. The present invention is based on magnetic resonance technology to detect dopamine, has good accuracy, sensitivity and selectivity, can accurately detect the signal of dopamine from the complicated system, and the interference of signals of other substances are well eliminated. Meanwhile, the present invention further has the advantages of simple operation and non-intervention, can be used for monitoring the content and distribution of the dopamine in a living body, and has important application value in the fields of biology and medicine.

TECHNICAL FIELD

The present invention relates to the technical field of magneticresonance, in particular to a method for selectively detecting dopaminemolecules based on magnetic resonance nuclear spin singlet, whichrealizes the selective observation of the ¹H NMR signals of dopamine.

BACKGROUND OF THE INVENTION

Dopamine is an important neurotransmitter in the brain. It participatesin many physiological and pathological activities of humans and mammals,especially plays a crucial role in movement regulation, learning andmemory, and drug addiction. Usually, the neurotransmitter such asdopamine is produced by the neurons those are also called dopaminergicneurons. An approach like the “returning satellite” is adopted to managethe released dopamine, ensuring that the amount of the released dopamineexactly fits the need of brain activity. At the same time, the dopaminetransporters are activated as the dopamine “recycling pump” to recyclethe released dopamine in a timely and appropriate amount. This not onlyachieves the purpose of regulating the concentration of extracellulardopamine and adapting to the needs of physiological activities, but alsoenables dopamine to be reused for energy saving and efficiencyenhancement. However, once the dopamine “recycling pump” system isdysfunctional, a variety of central nervous system diseases will occur,such as drug addiction.

At present, the detection methods of dopamine mainly include the flowinjection chemiluminescence, the high performance liquid chromatography,and the fluorescence method. These methods all require a complicatedpre-treatment process and cannot be used in the in-vivo detection ofdopamine in a living organism.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method forselectively detecting dopamine molecules based on magnetic resonancenuclear spin singlet. This method has good accuracy, sensitivity andselectivity, and can accurately detect dopamine signals from a systemwith complex components, and meanwhile can well eliminate theinterference of signals of the other substances.

The specific technical solution to achieve the purpose of the presentinvention is as follows:

Step A: Obtain the chemical shifts and J coupling values of the three ¹Hspins on the benzene ring of dopamine in the test sample;

Step B: According to the spin characteristics of dopamine, design apulse sequence that utilizes the nuclear spin singlet states to realizethe selection of dopamine signals;

Step C: By the designed pulse sequence, prepare the spin singlet statesof the three ¹H spin coupling system on the benzene ring of dopamine.During the existence of the spin singlets, the gradient field pulse isapplied to remove the other signals except the spin singlet in thesample, resulting in the targeting selection of dopamine signal.

The purpose of Step A is to obtain the nuclear spin couplingcharacteristics of dopamine molecules. It can usually be obtained byanalyzing the NMR spectrum of the sample. For example, FIG. 1 and FIG. 2show the ¹H spectra of dopamine. By analyzing the spectra, the chemicalshifts and J coupling values of the three ¹H spins on the benzene ringof the dopamine molecule can be obtained. The pulse sequence involved inthe present invention needs to be designed based on the characteristicsof the spin coupling system.

The pulse sequence designed in Step B should contain the module toprepare the spin singlets of the nuclear spin coupling system ofdopamine and the module to transform the spin singlets to the observablesignals, and the gradient field pulses during the evolution of thenuclear spin singlet to remove all of the other signals except thenuclear singlet. FIG. 3 shows a pulse sequence that can be used toprepare the spin singlets of dopamine. In this pulse sequence, firstly,a 90-degree hard pulse is applied along the x direction. After a delaytime of τ₁, a 180-degree hard pulse is applied along the x direction.Then, a delay time of (τ₁+τ₂) is applied, followed by a 90-degree hardpulse along the y direction, and then a delay time of τ₃ is applied.After τ₃, a delay time of τ₄ is given, followed by a 90-degree hardpulse along the y direction, and then a delay time of τ₅ is applied.

In this pulse sequence, the values of τ₁, t₂, τ₃ can affect theefficiency of the singlet preparation of the three ¹H spins on thebenzene ring of dopamine. In order to maximize the singlet preparationefficiency, optimization of τ₁, τ₂, τ₃ is required. τ₄ and τ₅ arerelated to the dopamine signals for the final detection. In order tomaximize the final detection signals, τ₄ and τ₅ need to be optimizedtoo. FIG. 4 shows the spectrum obtained using the pulse sequence of FIG.3. The sample is a dopamine aqueous solution sample.

The pulse sequence in FIG. 5 shows a way of applying the gradient fieldpulses during the evolution of the nuclear spin singlets. In FIG. 5,from the start of the pulse sequence to the end of τ₃ is the singletpreparation module. After this pulse sequence module, the first gradientfield pulse, g₁, is applied. Then there is the singlet evolution stage.In the singlet evolution stage, a decoupling pulse can be applied. Thepurpose of the decoupling pulse is to minimize the influence of theenvironmental factors on the spin singlets evolution. To better removeall of the other NMR signals except the spin singlets, a second gradientfield pulse g₂ can be applied after the decoupling period.

In Step C the three ¹H spin coupling system on the benzene ring ofdopamine is prepared into the nuclear spin singlets. There are severalmultiple spin systems in a dopamine molecule. The spin system formed bythe three ¹H on the benzene ring of dopamine can be effectively preparedfor the nuclear spin singlets. Ethylamine of dopamine also has a spincoupling system. However, the efficiency of the singlet preparation ofthis spin coupling system is poor.

The present invention also provides a method for selectively detectingdopamine molecules based on magnetic resonance nuclear (NMR) spinsinglet, comprises the following steps:

Steps 1: Put the D₂O aqueous solution with a mass fraction of 2%-5%dopamine in the magnetic resonance instrument, and apply a 90-degreehard pulse to the D₂O aqueous solution of dopamine, which makes the ¹Hsignals excited, obtain the ¹H spectrum of dopamine, and in turn thechemical shift of three ¹H on the benzene ring of dopamine and the Jcoupling values among the protons;

Steps 2: According to the pulse form of preparing the singlets from thetwo-spin system of the weakly coupled system, for the three-spin systemconsisting of three ¹H atoms on the benzene ring of dopamine, the pulseparameters for the preparation and detection of dopamine singlet in thethree-spin system are calculated based on chemical shifts and J couplingvalues obtained from Step 1 by using MATLAB. As a result, the pulsesequence to prepare the nuclear spin singlet states with the maximumefficiency of three ¹H atoms on the dopamine benzene ring can beobtained;

Steps 3: The complete pulse sequence is obtained by combining the pulseform to prepare the singlet of the two-spin system of the weakly coupledsystem and the pulse parameters required for the preparation anddetection of dopamine singlet in the three-spin system calculated inStep 2. The obtained pulse sequence can be applied to the dopamine D₂Oaqueous solution to prepare and detect the singlet of the three-spinsystem consisting of three ¹H on the benzene ring;

Steps 4: On the basis of preparation and detection of dopamine singletin Step 3, two gradient field pulses with different amplitudes and acontinuous wave (CW) decoupling pulse are applied between the pulses forthe singlet preparation and the pulses for the signal detection to forma new pulse sequence. The function of the new pulse sequence can bedivided into three parts: the first part is to obtain the singlet statesof the three ¹H on the benzene ring of dopamine; the second part is tokeep the singlet states of three ¹H on the benzene ring of the dopamineand filter the other non-single state signals because the nuclear spinsinglet state is not affected by the gradient field pulses and the CWdecoupling pulse; the third part is to detect the singlet states ofthree ¹H on the benzene ring; in the end, only the three ¹H signals onthe benzene ring are kept, achieving the purpose of selective signalfiltering. In this process, it is necessary to continuously optimize thetime of the CW pulse to achieve the best filtering efficiency.

As for the ¹H spectrum of dopamine described in Step 1, the threesignals on the left side represent the three ¹H signals on the benzenering, the single peak in the middle is the water signal, and the ethylsignal of dopamine is on the right side; the J coupling values andchemical shifts between the three ¹H atoms on the benzene ring ofdopamine are obtained from the ¹H spectrum of dopamine.

Step 2 is as follows: firstly, a 90-degree hard pulse is applied along xdirection, after a delay time of τ₁, a 180-degree hard pulse is appliedalong the x direction; then, a delay time of (τ₁+τ₂) is applied,followed by a 90-degree hard pulse along the y direction, and then adelay time of τ₂/2 namely τ₃ is applied; the function of this pulse isto prepare the singlet states of three ¹H on the benzene ring ofdopamine, which is called preparation pulse; because the singlet statescannot be directly detected, another pulse is needed to detect thesinglets of the three ¹H of three-spin system on the benzene ring ofdopamine, which is called detection pulse; the form of detection pulseis as follows, after a delay time of τ₄, followed by a 90-degree hardpulse along the y direction and then a delay time of τ₅ is applied;next, take ADC sample immediately until the sampling signal decay iscompleted; in this process, the values of τ₁ and τ₂ impact theefficiency of the singlet states consisting of three ¹H atoms on thebenzene ring of dopamine; in order to maximize the efficiency of thesinglet states, MATLAB software is used to calculate the values of τ₁and τ₂; first of all, 64 basic operators of the three-spin system areconstructed in the MATLAB script, and then the Hamiltonian of thethree-spin system consisting of three ¹H atoms on the benzene ring ofdopamine is written; finally, the operating operators corresponding tothe 90-degree hard pulse and the 180-degree hard pulse are obtained;then, the system is continuously evolving from the thermal equilibriumsignals under the operating operator and Hamiltonian of the hard pulse,and the evolution time τ₁ and τ₂ are continuously optimized to maximizethe singlet states preparation efficiency; similarly, on the basis ofgenerating singlet states, evolution time τ₄ and τ₅ are optimized tomaximize the singlet states detection efficiency; finally, the completepulse to prepare and detect three-spin system singlet states of dopamineis obtained by combining the pulse form of the two-spin system singletof the weakly coupled system and the calculated pulse parameters.

As described in Step 3, the singlet state of the three-spin systemconsisting of three ¹H atoms on the benzene ring of dopamine is preparedand detected. Specifically, firstly, the complete pulse obtained in Step2 is written into the computer by the NMR instrument language; secondly,a D₂O aqueous solution of dopamine is put into the magnetic resonanceinstrument, and then the field-locking, field-shimming, matching, andtuning are performed; finally, the radio frequency center of thetransmitter is set to the three ¹H on the benzene ring of dopamine, andthe complete pulse written into the computer is applied to prepare anddetect the singlet states of the dopamine.

As described in Step 4, the CW decoupling pulse and two gradient fieldpulses with different amplitudes are applied between the pulses for thesinglet preparation and the pulses for the signal detection. This formsa new pulse module. Specifically, the duration of the CW pulse isbetween 50 ms and 1 s; the amplitude varies from 1 watt to 15 watts; theduration of each of the two gradient field pulses with differentamplitudes along the z direction is between 1 ms and 5 ms, with theamplitude varying from 5 to 10 Gauss/cm. This new pulse module includingthe CW decoupling pulse and the two gradient field pulses with differentamplitudes, is written into the computer by the NMR instrument language.Then, the routine procedures such as field-locking, field-shimming,matching, and tuning are performed; finally, the radio frequency centerof the transmitter is set to the three ¹H on the dopamine benzene ring,and the new pulse sequence which has been written into the computerincluding the CW decoupling pulse and the two gradient field pulses withdifferent amplitudes is applied to prepare and detect the singlet statesof dopamine.

Compared with the existing technology, the beneficial effects of thepresent invention are as follows:

The invention has good accuracy, sensitivity and selectivity and canaccurately detect the dopamine signals from a system containing complexcomponents. At the same time, it can eliminate the interference of thesignals of the other substances in the sample. This invention also hasthe advantages of simple operation and non-intervention. It can be usedto monitor the amount and distribution of dopamine in-vivo, and hasimportant applications in the fields of biology and medicine.

Compared with the existing technologies, the present invention has thefollowing advantages:

(1) The existing magnetic resonance technology cannot achieve selectiveobservation of dopamine signals while suppressing all other signals inthe sample;

(2) It can be applied to living organisms without the need to injectmolecular probes into the organism, and can detect dopamine molecules inreal-time and in vivo without damaging tissue cells;

(3) There is no need to separate the dopamine molecules in the testedsample, and it can be applied to the detection of dopamine in themixture samples.

(4) There is no dependence on the field strength, as long as the Jcoupling values and the chemical shifts of the three ¹H spins on thebenzene ring of dopamine are obtained, the required parameters in thepulse sequence can be calculated by MATLAB, and the selectiveobservation of dopamine can be realized.

(5) The method is simple. There is no need to damage the dopaminemolecule, and selective detection of the dopamine molecules can beachieved by using the specific pulse sequence in an NMR instrument.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the molecular structure of dopamine and the ¹H NMR spectrum ofthe dopamine aqueous solution sample.

FIG. 2 is the ¹H NMR spectrum of the three ¹H spins on the benzene ringof dopamine aqueous solution sample to prepare the dopamine singlet withthe spectrum range from 6.61 ppm to 6.89 ppm.

FIG. 3 is the scheme of the pulse sequence for the singlet preparationof dopamine. The black rectangle represents a 90-degree hard pulse, andthe white square represents a 180-degree hard pulse. x and y representthe phases of the radio frequency (RF) pulses. τ₁-τ₅ is the time for thespin evolution. In FIG. 3, the pulse sequence module from the start ofthe pulse sequence to the end of τ₃ is the preparation module of thespin singlets of the nuclear spin coupling system of dopamine. The pulsesequence module from the beginning of τ₄ to the end of τ₅ is the moduleto transform the spin singlets to the observable signals.

FIG. 4 is the ¹H NMR spectrum obtained through preparing and detectingthe singlet states of the dopamine in Example 1. The pulse sequence inFIG. 3 is used to acquire the spectrum.

FIG. 5 is the scheme of the pulse after adding the gradient field pulseand the high power CW decoupling pulse on the basis of preparing anddetecting singlet. The selected pulse sequence of dopamine signals isrealized by using the nuclear spin singlet. The black rectanglerepresents a 90-degree pulse and the white square represents a180-degree pulse. x and y represent the phases of the radio frequency(RF) pulses. τ₅ is the time for the spin evolution. τ_(m) is thedecoupling pulse time, and g₁ and g₂ represent the gradient fieldpulses.

FIG. 6 is the ¹H NMR spectrum of the dopamine aqueous solution sample.In Example 1 of the invention, after applying the gradient field pulsesand high power CW decoupling pulse on the basis of the original pulsesequence, the ¹H NMR spectrum is obtained by preparing and detecting thespin singlets of dopamine; the pulse sequence in FIG. 5 is used toacquire the spectrum. The water signal in the spectrum was significantlysuppressed.

FIG. 7 is the ¹H NMR spectrum of a mixture aqueous solution sample. Thesolute in this sample is a mixture consisting of dopamine, creatine,inositol, and glutamine.

FIG. 8 is the ¹H NMR spectrum of a mixture aqueous solution sample. Thesolute in this sample is a mixture consisting of dopamine, creatine,inositol, and glutamine. After using the pulse sequence which has addingthe gradient field pulses and the high power CW decoupling pulse on thebasis of the original pulse sequence, the ¹H NMR spectrum can beobtained by preparing and detecting the spin singlets of dopamine in themixture aqueous solution sample; The pulse sequence in FIG. 5 is used toacquire the spectrum. In this spectrum, except the signals from thebenzene ring of dopamine, all other signals are greatly suppressed.

FIG. 9 is the ¹H NMR spectrum is obtained by preparing and detecting thespin singlets of dopamine in the complex system in Example 2 of thisinvention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention will be further described in detail with thereference to the following specific examples and figures.

Example 1

NMR Instrument: Bruker AVANCE III 500 NMR spectroscopy.The example was carried out as follows:

Step A: Analyze the ¹H NMR spectrum of dopamine and make the signalassignment. In the ¹H NMR spectrum of dopamine (see FIG. 1 and FIG. 2),if setting the center frequency between peaks 1 and 3 to zero, theresonance frequencies and J coupling values of peaks 1, 2 and 3 (i.e.,the characteristics of the three ¹H spin system of dopamine) are:

ω₁=36.5 Hz,ω₂=−7.8 Hz,ω₃=−36.5 Hz;

J ₁₂=0 Hz,J ₁₃=8.14 Hz,J ₂₃=2.18 Hz.

Step B: Design a pulse sequence that can be used to selectively detectthe signals of dopamine molecules based on the nuclear spin singlets.According to the characteristics of the spin system of dopamine, thepulse sequence in FIG. 5 was designed. The core steps and ideas of thedesign of the pulse sequence are as follows: Firstly, the Pauli operatoris used to construct the base operator of the three-spin system. Thenaccording to the characteristics of the three ¹H spin system of dopamineobtained in Step A (i.e., the three ¹H resonance frequencies and the Jcoupling values), the spin Hamiltonians and the evolution operators ofthe three-spin system are as follows:

Ĥ=ω ₁ Î _(1z)+ω₂ Î _(2z)+ω₃ Î _(3z)+2πJ ₁₂ Î _(1z) Î _(2z)+2πJ ₁₃ Î_(1z) Î _(3z)+2πÎ _(2z) Î _(3z)

û(τ)=e ^(−iĤτ)

û*(τ)=e ^(iĤτ)

wherein, ω₁, ω₂, ω₃ represent the resonance frequencies of the three ¹Hspins on the benzene ring of dopamine. Î_(1z),Î_(2z),Î_(3z) are the baseoperators along the z axis of the three-spin system. τ is the evolutiontime, û(τ) is the evolution operator after τ time, û*(τ) is thetransposed complex conjugate of ŷ(τ), and Ĥ is the spin systemHamiltonian.

The operator of the φ degree hard pulse along the x direction is asfollows:

û _(x)(φ)=e ^(−i*(Î) ^(1x) ^(+Î) ^(2x) ^(+Î) ^(3x) ^()*φ)

û* _(x)(φ)=e ^(i*(Î) ^(1x) ^(+Î) ^(2x) ^(+Î) ^(3x) ^()*φ)

wherein, Î_(1x),Î_(2x),Î_(3x) are the base operators along the x axis ofthe three-spin system, and û*_(x)(φ) is the transposed complex conjugateof û_(x)(φ).

The three-spin system is in the thermal equilibrium at room temperature,and thus the density operator is as follows:

{circumflex over (ρ)}₀ =Î _(1z) +Î _(2z) +Î _(3z)

The evolution process of the density operator corresponding to the pulsesequence in FIG. 5 is as follows:1. After applying a 90-degree hard pulse along the x direction, thedensity operator can be written as:

${\hat{\rho}}_{1} = {{{\hat{u}}_{x}\left( \frac{\pi}{2} \right)}{\hat{\rho}}_{0}{{\hat{u}}_{x}^{*}\left( \frac{\pi}{2} \right)}}$

2. After the evolution through time of τ₁, the density operator becomes:

{circumflex over (ρ)}₂ =û(τ₁){circumflex over (ρ)}₁ û*(τ₁)

3. After applying a 180-degree hard pulse along the x direction, thedensity operator becomes:

{circumflex over (ρ)}₃ =û _(x)(π){circumflex over (ρ)}₂ û* _(x)(π)

4. After the evolution through time of τ₁+τ₂, the density operatorbecomes:

{circumflex over (ρ)}₄ =û(τ₁+τ₂){circumflex over (ρ)}₃ û*(τ₁+τ₂)

5. After applying a 90-degree hard pulse along the y direction, thedensity operator becomes:

${\hat{\rho}}_{5} = {{{\hat{u}}_{y}\left( \frac{\pi}{2} \right)}{\hat{\rho}}_{4}{{\hat{u}}_{y}^{*}\left( \frac{\pi}{2} \right)}}$

6. After the evolution through time of τ₃, the density operator becomes:

{circumflex over (ρ)}₆ =û(τ₃){circumflex over (p)} ₅ û*(τ₃)

After the evolution under the operation operators of Hamilton and thehard pulse, the target operator of the single state is:

$\hat{Q} = {{\frac{1}{\sqrt{3}}\left( {{{\overset{\hat{}}{I}}_{1x}{\overset{\hat{}}{I}}_{3x}} + {{\overset{\hat{}}{I}}_{1y}{\overset{\hat{}}{I}}_{3y}} + {{\overset{\hat{}}{I}}_{1z}{\overset{\hat{}}{I}}_{3z}}} \right)} + {\frac{1}{\sqrt{3}}\left( {{{\overset{\hat{}}{I}}_{2x}{\overset{\hat{}}{I}}_{3x}} + {{\overset{\hat{}}{I}}_{2y}{\overset{\hat{}}{I}}_{3y}} + {{\overset{\hat{}}{I}}_{2z}{\overset{\hat{}}{I}}_{3z}}} \right)}}$

The trace of the product of {circumflex over (ρ)}₆ and the targetoperator {circumflex over (Q)} yields the singlet conversion efficiency,S:

S=trace({circumflex over (Q)}{circumflex over (ρ)} ₆)

To maximize the singlet conversion efficiency, τ₁, τ₂, and τ₃ need to beoptimized.

7. After the evolution through time of τ₃, the gradient field pulse, g₁,is applied to remove all other signals except the spin singlets in thesample. The spin singlets are not affected by the gradient field pulse,g₁.8. After the gradient field pulse, g₁, a decoupling pulse with a periodof τ_(m) is applied to preserve the spin singlets in the sample.9. After the decoupling pulse, the gradient field pulse, g₂, is appliedto further suppress all other signals except the spin singlets in thesample.

Because the spin singlets cannot be directly detected in the NMRinstrument, it needs to be converted into the observable signals. Thesignal evolution of the system after the gradient field pulse, g₂, is asfollows:

10. After the evolution through time of τ₄, the density operatorbecomes:

{circumflex over (ρ)}₇ =û(τ₄){circumflex over (Q)}û*(τ₄)

11. After applying a 90-degree hard pulse along the y direction, thedensity operator becomes:

${\hat{\rho}}_{8} = {{{\hat{u}}_{y}\left( \frac{\pi}{2} \right)}{\hat{\rho}}_{7}{{\hat{u}}_{y}^{*}\left( \frac{\pi}{2} \right)}}$

12. After the evolution through time of τ₅, the density operatorbecomes:

{circumflex over (ρ)}₉ =û(τ₅){circumflex over (Q)}û*(τ₅)

If using the quantum state after the singlet preparation as the initialstate, to have the best optimized singlet preparation efficiency, thetarget quantum state is:

{circumflex over (P)}=½(Î _(1x) Î _(3z) +Î _(1z) Î _(3x))+½(Î _(2x) Î_(3z) +Î _(3x) Î _(2z))

The trace of the product of {circumflex over (ρ)}₉ and the targetquantum state {circumflex over (P)} yields the projection of the finalstate on the target quantum state:

R=trace({circumflex over (P)}{circumflex over (ρ)} ₉)

wherein, R represents the observable signal transformed from the spinsinglets. In order to achieve the maximum value of R, τ₄ and τ₅ need tobe optimized.

Step C: Use the pulse sequence designed in Step B to selectively detectthe signals of dopamine. The experimental parameters need to beoptimized. The optimized experimental parameters in this experiment areas follows: τ₁=30.9 ms, τ₂=6.8 ms, τ₃=3.4 ms, τ₄=6.8 ms, τ₅=3.3 ms. Thegradient field pulse, g₁, is applied along the z direction, the pulselength is 1 ms and the amplitude is 5 Gauss/cm. The direction, length,and power of the gradient pulse, g₂, is the same as g₁. A CW pulse isused as the decoupling pulse. The decoupling time is 100 ms, and thedecoupling amplitude is 3 watts. The NMR spectrum in FIG. 6 is obtainedby using the above parameters in the experiment. The sample is adopamine deuterium aqueous solution (i.e. dopamine dissolved in D₂O).

This embodiment comprises the following steps:

The instrument used in this embodiment is Bruker AVANCE III 500 NMRspectroscopy.

The specific steps from the singlet preparation of dopamine to therealization of the filtering of dopamine signals are as follows:

Step 1: This embodiment is a system in which dopamine is dissolved inD₂O. Preparing an aqueous solution of dopamine with a mass fraction of3%. In the NMR experiment, a 90-degree hard pulse is applied, obtainingthe signal peaks of the dopamine ¹H spectrum shown in FIG. 1. Amongthem, the peaks of three protons on the dopamine benzene ring are at thefar left, and the water peak is at the middle of the spectrum, thesignal on the right is the ethyl signal of the dopamine molecule.Selecting the three ¹H peaks of the benzene ring in the dopaminestructure as the target peaks (The labeled peaks 1, 2, and 3 on the ¹Hspectrum is shown in FIG. 2). After setting the center frequency betweenpeaks 1 and 3 to zero, the J coupling values between the protons of thesystem and their corresponding chemical shifts read as:

ω₁=36.5 Hz,ω₂=−7.8 Hz,ω₃=−36.5 Hz;

J ₁₂=0 HZ,J ₁₃=8.14 Hz,J ₂₃=2.18 Hz.

Step 2: Firstly, the Pauli operator is used to construct the baseoperator of the three-spin system. Then according to the chemical shiftsand the J coupling values of the three ¹H spin system of dopamineobtained in Step 1, the spin Hamiltonians and the evolution operators ofthe three-spin system are as follows:

Ĥ=ω ₁ Î _(1z)+ω₃ Î _(2z)+ω₃ Î _(3z)+2πJ ₁₂ Î _(1z) Î _(2z)+2πJ ₁₃ Î_(1z) Î _(3z)+2πJ ₂₃ Î _(2z) Î _(3z)

û(τ)=e ^(−iĤτ)

û*(τ)=e ^(iĤτ)

Wherein, ω₁, ω₂, ω₃ represent the values of the chemical shift of thethree 41 spins on the benzene ring of dopamine respectively.Î_(1z),Î_(2z),Î_(3z) are the base operators along the z axis of thethree-spin system; T is the evolution time, û(τ) is the evolutionoperator after τ time, û*(τ) is the transposed complex conjugate ofû(t), and Ĥ is the Hamiltonian of the spin system.

Shown as an example, the operator of the 90-degree hard pulse along thex direction is as follows:

${{\hat{u}}_{x}\left( \frac{\pi}{2} \right)} = e^{{- i}*{({{{\overset{\hat{}}{I}}_{1x}{\overset{\hat{}}{I}}_{2x}} + {\overset{\hat{}}{I}}_{3x}})}*\frac{\pi}{2}}$${{\hat{u}}_{x}^{*}\left( \frac{\pi}{2} \right)} = e^{i*{({{{\overset{\hat{}}{I}}_{1x}{\overset{\hat{}}{I}}_{2x}} + {\overset{\hat{}}{I}}_{3x}})}*\frac{\pi}{2}}$

Wherein, Î_(1x),Î_(2x),Î_(3x) are the base operators along the x axis ofthe three-spin system, and

${\hat{u}}_{x}^{*}\left( \frac{\pi}{2} \right)$

is the transposed complex conjugate of

${{\hat{u}}_{x}\left( \frac{\pi}{2} \right)}.$

The three-spin system is in the thermal equilibrium at room temperature,and thus the density operator is as follows:

{circumflex over (ρ)}₀ =Î _(1z) +Î _(2z) +Î _(3z)

The pulse form of the two-spin system singlet prepared in the weaklycoupled system is as shown in FIG. 3b

1. After applying a 90-degree hard pulse along the x direction, thedensity operator can be written as:

${\hat{\rho}}_{1} = {{{\hat{u}}_{x}\left( \frac{\pi}{2} \right)}{\hat{\rho}}_{0}{{\hat{u}}_{x}^{*}\left( \frac{\pi}{2} \right)}}$

2. After the evolution through time of τ₁, the density operator becomes:

{circumflex over (ρ)}₂ =û(τ₁){circumflex over (ρ)}₁ û*(τ₁)

3. After applying a 180-degree hard pulse along the x direction, thedensity operator becomes:

{circumflex over (ρ)}₃ =û _(x)(π){circumflex over (ρ)}₂ û* _(x)(π)

4. After the evolution through time of τ₁+τ₂, the density operatorbecomes:

{circumflex over (ρ)}₄ =û(τ₁+τ₂){circumflex over (p)} ₃ û*(τ₁+τ₂)

5. After applying a 90-degree hard pulse along the y direction, thedensity operator becomes:

${\hat{\rho}}_{5} = {{{\hat{u}}_{y}\left( \frac{\pi}{2} \right)}{\hat{\rho}}_{4}{{\hat{u}}_{y}^{*}\left( \frac{\pi}{2} \right)}}$

6. After the evolution through time of τ₃, the density operator becomes:

{circumflex over (ρ)}₆={circumflex over (u)}(τ₃){circumflex over(ρ)}₅{circumflex over (u)}*(τ₃)

Wherein,

$\tau_{3} = \frac{\tau_{2}}{2}$

The function of this pulse is to prepare the singlet state of thethree-spin system consisting of three ¹H on the benzene ring ofdopamine. It can be named as the preparation pulse for short.

After the evolution under the operation operators of Hamilton and hardpulse, the targeted operator of the singlet state is:

$\hat{Q} = {{\frac{1}{\sqrt{3}}\left( {{{\overset{\hat{}}{I}}_{1x}{\overset{\hat{}}{I}}_{3x}} + {{\overset{\hat{}}{I}}_{1y}{\overset{\hat{}}{I}}_{3y}} + {{\overset{\hat{}}{I}}_{1z}{\overset{\hat{}}{I}}_{3z}}} \right)} + {\frac{1}{\sqrt{3}}\left( {{{\overset{\hat{}}{I}}_{2x}{\overset{\hat{}}{I}}_{3x}} + {{\overset{\hat{}}{I}}_{2y}{\overset{\hat{}}{I}}_{3y}} + {{\overset{\hat{}}{I}}_{2z}{\overset{\hat{}}{I}}_{3z}}} \right)}}$

After the evolution under the operation operators of hard pulse andHamilton, the trace of the product of {circumflex over (ρ)}₆ and thetarget operator {circumflex over (Q)} yields the projection on thesinglet state:

S=trace({circumflex over (Q)}{circumflex over (ρ)} ₆)

Wherein, S represents the conversion efficiency of the singlet state. Inorder to maximize the singlet state conversion efficiency, it is neededto use MATLAB to perform a simulation to obtain the appropriateparameters. Firstly, writing the above formulas into MATLAB scriptsrespectively; then continuously optimize the values of τ₁, τ₂ throughprogramming, so that the singlet state conversion efficiency (theabsolute value of S) reaches the optimal value. After optimizing,τ₁=30.9 ms, τ₂=6.8 ms, and the singlet efficiency can reach the optimalvalue.

Because the single state is not a single quantum signal, and the spinsinglet cannot be directly detected in the NMR instrument. It isnecessary to apply the detection pulse in order to detect the singletstate signals. The detection pulse form is as follows:

1. After the evolution through time of τ₄, the density operator becomes:

{circumflex over (ρ)}₇ =û(τ₄){circumflex over (Q)}û*(τ₄)

2. After applying a 90-degree hard pulse along the y direction, thedensity operator becomes:

${\hat{\rho}}_{8} = {{{\hat{u}}_{y}\left( \frac{\pi}{2} \right)}{\hat{\rho}}_{7}{{\hat{u}}_{y}^{*}\left( \frac{\pi}{2} \right)}}$

3. After the evolution through time of τ₅, the density operator becomes:

{circumflex over (ρ)}₉ =û(τ₅){circumflex over (Q)}û*(τ₅)

If using the quantum state after the singlet preparation as the initialstate, to have the best-optimized singlet preparation efficiency, thetarget quantum state is:

{circumflex over (P)}=½({circumflex over (I)}_(1x){circumflex over(I)}_(3z)+{circumflex over (I)}_(1z){circumflex over(I)}_(3x))+½({circumflex over (I)}_(2x){circumflex over(I)}_(3z)+Î_(3x){circumflex over (I)}_(2z))

Similarly, after the detection pulse, the trace of the product of{circumflex over (ρ)}₉ and the target quantum state {circumflex over(P)} yields the projection of the final state on the target quantumstate:

R=trace({circumflex over (P)}{circumflex over (ρ)} ₉)

Wherein, R represents the conversion efficiency of the target quantumstate. In order to achieve the maximum value of R, τ₄ and τ₅ need to becontinuously optimized by MATLAB to maximize the absolute value of R.After optimizing, τ₄=3.3 ms,τ₅=6.8 ms.

After calculation, the complete pulse sequence to prepare and detect thethree-spin system of dopamine is obtained by combining the pulse form ofthe two-spin system singlet under the weakly coupled system and thecalculated pulse parameters.

Step 3: The complete pulse obtained in Step 2 is written into thecomputer by NMR instrument language; then, a D₂O aqueous solution ofdopamine is put into the NMR instrument, and then the field-locking,field-shimming, matching, and tuning are performed; finally, the radiofrequency center of the transmitter is set to the three ¹H on thebenzene ring of dopamine, and the complete pulse written into thecomputer is applied to prepare and detect the singlet states ofdopamine. The detected NMR spectrum of dopamine is shown in FIG. 4. Thethree signals on the left are from the three protons on the dopaminebenzene ring, and the right is the water signal in deuterium water. Itcan be seen that after the singlet preparation and detection, the signalintensity of water in deuterium water is still far greater than thesignal of dopamine, and selective filtering has not been achieved.Therefore, it is needed to be improved on the basis of preparing singlestates.

The pulse sequence for preparing the spin singlets of dopamine can bedesigned according to actual needs, and different preparation singletpulse sequences can be used to realize the singlet preparation ofdopamine.

Step 4: Based on the preparation and detection of singlet state in Step3, a new high power CW pulse and two gradient field pulses withdifferent amplitudes are applied between the pulses for the singletpreparation and the pulses for the signal detection, these pulses formare shown in FIG. 5. The pulses before b are the preparation module ofthe dopamine singlet. The CW pulse and two gradient field pulses withdifferent amplitudes are applied in the period between b and c. Thepulses after c are the module to detect the singlet of dopamine.Wherein, the duration of the CW pulse is 100 ms, the amplitude is 3watts. Two gradient field pulses with different amplitudes require aduration of 1 ms, an amplitude of 5 Gauss/cm along the z direction. Inthe process, the duration and amplitude of CW are needed to be optimizedin order to achieve the best filtering effect, that is, filtering outnon-singlet signal components while keeping the three ¹H signals fromthe benzene ring of dopamine. The experimental result is shown in FIG.6. The three signals on the left are from the three ¹H on the dopaminering, and the signal on the right is the water signal in deuteriumwater. The signal intensity of water in deuterium water is much smallerthan the signal of dopamine molecules after applying the CW decouplingpulse and two gradient field pulses with different amplitudes betweenthe preparation module of the spin singlets and the module of spinsinglet detection. This achieves selectively filtering.

Example 2

NMR Instrument: Bruker AVANCE III 500 NMR spectroscopy.The example was carried out as follows:

Step A: Prepare an aqueous solution of dopamine, creatine, inositol, andglutamine with a mass fraction of 1.5% for each solute. D₂O instead ofH₂O is used to prepare the solution. The ¹H NMR spectrum of the sampleis shown in FIG. 7. Analyze the ¹H NMR spectrum and make the signalassignment. The leftmost signals are from the three protons on thebenzene ring of dopamine. The middle signal at 4.7 ppm is assigned tothe water (HDO). The ¹H signals in the right side of the spectrum arefrom the ethyl group of dopamine, and the groups of creatine, inositol,and glutamine. If setting the center frequency between peaks 1 and 3 tozero (see FIG. 7), the resonance frequencies and J coupling values ofpeaks 1, 2 and 3 are:

ω₁=36.5 Hz,ω₂=−7.8 Hz,ω₃=−36.5 Hz;

J ₁₂=0 Hz,J ₁₃=8.14 Hz,J ₂₃=2.18 Hz.

Step B: Because the resonance frequencies and J coupling values of peaks1, 2 and 3 have not been changed in the complex system, and the spinHamiltonian thus remains unchanged too. The pulse sequence in FIG. 5 wasdirectly used in the experiments in this example.

Step C: This step is similar to Step C in Example 1. The parameters usedin the experiments are the same as those used in Example 1. The spectrumin FIG. 8 was obtained.

This embodiment comprises the following steps:

The instrument used in this embodiment is Bruker AVANCE III 500 NMRspectroscopy.

The specific steps from the preparation of the spin singlet of dopamineto the realization of the signal filtering are as follows:

Steps 1: Prepare an aqueous solution of dopamine, creatine, inositol,and glutamine with a mass fraction of 1.5% for each solute. Put thesolution sample into an NMR instrument. After applying a 90-degree hardpulse, the signals of dopamine as shown in the ¹H spectrum in FIG. 7 canbe obtained. In the spectrum, the leftmost signals are the three ¹H onthe benzene ring of dopamine. The middle signal at 4.7 ppm is assignedto the water (HDO). The ¹H signals in the right side of the spectrum arefrom the ethyl of dopamine, and the groups of creatine, inositol andglutamine. The J coupling values between the protons of the system andthe corresponding chemical shifts read as:

ω₁=36.5 Hz,ω₂=−7.8 Hz,ω₃=−36.5 Hz;

J ₁₂=0 Hz,J ₁₃=8.14 Hz,J ₂₃=2.18 Hz

Step 2: Because the chemical shifts and J coupling values of peaks 1, 2and 3 have not been changed in the complex system, and the spinHamiltonian thus remains unchanged too. The complete pulse sequence ofStep 2 of Example 1 can be directly used, which is composed of the pulsesequence for preparing the singlet state of the two-spin system and thecalculated pulse parameters under the weakly coupled system.

Step 3: Similar to Step 3 in Example 1, applying the pulse, which hasbeen written into the computer, to prepare and detect the spin singletof dopamine. The detected dopamine NMR spectrum is shown in FIG. 9. Theintensity of the signals of the three protons from the benzene ring ofthe dopamine molecule at the far left barely changes. But the phase ofthe signals changes. The signal at 4.7 ppm is the water signal, whichhas been suppressed greatly. The ¹H signals in the right side of thespectrum are from the ethyl of dopamine, the groups of creatine,inositol and glutamine. The intensity of these signals changed. Butcompared with that of dopamine, these signals are still higher thanthose of dopamine. In general, compared with the ¹H NMR spectrum (FIG.7), the intensity of the other non-singlet signals in the spectrum issuppressed to a certain extent, but the effect is not obvious except forthe water signal.

Step 4: Based on the preparation and detection of singlet state in Step3, a new high power CW pulse and two gradient field pulses withdifferent amplitudes are added between the preparation module of thespin singlets and the module of spin singlet detection. These pulses areshown in FIG. 5. The pulses before b belong to the preparation module ofthe spin singlets of dopamine. The pulses between b and c are the newlyapplied CW pulse and the two gradient field pulses with differentamplitudes. The pulses after c are the module of spin singlet detection.Wherein, the duration of the CW pulse is 100 ms, the amplitude is 3watts. Two gradient field pulses with different amplitudes require aduration time of 1 ms, with an amplitude of 5 Gauss/cm along the zdirection. The results are shown in FIG. 8. The ¹H signals which arefrom the benzene ring of dopamine at the far left of the spectrum show aclear decay, and meanwhile, the phases of the signals change. The signalat 4.7 ppm is the water signal and the signal intensity is almost 0.Calculation shows that compared with the water signal in the ¹Hspectrum, the height of the residual water signal is less than 0.1%. The¹H signals in the right side of the spectrum are from the ethyl ofdopamine, the groups of creatine, inositol and glutamine. Theintensities of these signals are obviously changed. Comparing with the¹H signals which are from the benzene ring of dopamine, theirintensities are almost negligible. In summary, the other non-singletsignals in the spectrum are almost completely suppressed in the signalintensity compared with the ¹H spectrum (FIG. 9). In particular, thesignal of water is almost attenuated to 0 in intensity, and only thesignals from the singlets of dopamine are retained, realizing theselective detection of the dopamine signals in this mixed system.

The present invention discloses a method for selectively detectingdopamine molecules based on the magnetic resonance nuclear spin singlet.The method utilizes the nuclear spin singlet of the three ¹H spins onthe benzene ring of dopamine to realize the selective detection ofdopamine signals in complex systems. The present invention detectingdopamine by the magnetic resonance technique, has good accuracy,sensitivity and selectivity, and can accurately detect dopamine signalsfrom a system having complex components, and meanwhile can welleliminate the interference of signals from the other substances. At thesame time, the present invention has the advantages of simple operationand non-intervention, and thus can be used to monitor the amount anddistribution of the dopamine molecules in a living body. Thus thisinvention will have important applications in the fields of biology andmedicine.

The content described above is only the preferred embodiments of thepresent invention. The skilled persons in the field may modify orequivalently make some substitution within the spirit of the presentinvention. All changes made in accordance with the spirit of the presentinvention should fall within the scope of protection claimed by thepresent invention.

1. A method for selectively detecting dopamine molecules based onmagnetic resonance nuclear (NMR) spin singlet comprising: step A: obtainthe chemical shifts and J coupling values of the three ¹H spins on thebenzene ring of dopamine in the test sample; step B: according to thespin characteristics of dopamine, design a pulse sequence that utilizesthe nuclear spin singlet states to realize the selection of dopaminesignals; step C: by the designed pulse sequence, prepare the spinsinglet states of the three ¹H spin coupling system on the benzene ringof dopamine; during the existence of the spin singlets, the gradientfield pulse is applied to remove the other signals except the spinsinglet in the sample, resulting in the targeting selection of dopaminesignal.
 2. The method of claim 1, wherein the purpose of step A is toobtain the nuclear spin coupling characteristics of dopamine moleculesby analyzing the NMR spectrum of the test sample.
 3. The method of claim1, wherein the designed pulse sequence of step B contains thepreparation module of the spin singlets of the nuclear spin couplingsystem of dopamine, and the module to transform the spin singlets to theobservable signals.
 4. The method of claim 3, wherein, in the module,firstly a 90-degree hard pulse is applied along the x direction; after adelay time of τ₁, a 180-degree hard pulse is applied along the xdirection; then, a delay time of (τ₁+τ₂) is applied, followed by a90-degree hard pulse along the y direction, and then a delay time of τ₃is applied; from the start of the pulse sequence to the end of τ₃, thispulse sequence module is used to prepare the spin singlets of the three¹H spin system on the benzene ring of dopamine; after τ₃, a delay timeof τ₄ is given, followed by a 90-degree hard pulse along the y directionand then a delay time of τ₅ is applied; this pulse sequence module isused to transform the spin singlets into the observable signals; in thedesigned pulse sequence, the values of τ₁, τ₂, τ₃ can affect theefficiency of the singlet preparation of the three ¹H spins on thebenzene ring of dopamine; in order to maximize the singlet preparationefficiency, optimization of τ₁, τ₂, τ₃ is required; τ₄ and τ₅ arerelated to the dopamine signals for the final detection; in order tomaximize the final detection signals, τ₄ and τ₅ need to be optimizedtoo.
 5. The method of claim 1, wherein the designed pulse sequence ofstep B comprises the pulse sequence module in which the gradient fieldpulses are applied during the evolution of the nuclear spin singlets toremove all other NMR signals except the nuclear spin singlets; from thestart of the pulse sequence to the end of τ₃ is the pulse sequencemodule for the singlet preparation; after this pulse sequence module,the first gradient field pulse, g₁, is applied, then there is thesinglet evolution stage; in the singlet evolution stage, a decouplingpulse can be applied; the purpose of the decoupling pulse is to minimizethe influence of the environmental factors on the spin singletsevolution; to better remove all of the other NMR signals except the spinsinglets, a second gradient field pulse g₂ can be applied after thedecoupling period.
 6. The method of claim 1, wherein the methodspecifically comprises the following steps: steps 1: put the D₂O aqueoussolution with a mass fraction of 2%-5% dopamine in the magneticresonance instrument, and apply a 90-degree hard pulse to the D₂Oaqueous solution of dopamine, which makes the ¹H signals excited, thenobtain the ¹H spectrum of dopamine, and in turn the chemical shifts ofthree ¹H on the benzene ring of dopamine and the J coupling values amongthe protons; steps 2: according to the pulse form of preparing thesinglets from the two-spin system of the weakly coupled system, for thethree-spin system consisting of three ¹H atoms on the benzene ring ofdopamine, the pulse parameters for the preparation and detection ofdopamine singlet in the three-spin system are calculated based onchemical shifts and J coupling values obtained from step 1 by usingMATLAB; as a result, the pulse sequence to prepare the nuclear spinsinglet states of three ¹H atoms on the dopamine benzene ring with themaximum efficiency can be obtained; steps 3: the complete pulse sequenceis obtained by combining the pulse form to prepare the singlet of thetwo-spin system of the weakly coupled system and the pulse parametersrequired for the preparation and detection of dopamine singlet in thethree-spin system calculated in step 2, which is applied to the dopamineD₂O aqueous solution to prepare and detect the singlet of the three-spinsystem consisting of three ¹H on the benzene ring; steps 4: on the basisof preparation and detection of dopamine singlet in step 3, two gradientfield pulses with different amplitudes and a continuous wave (CW)decoupling pulse are applied between the pulses for the singletpreparation and the pulses for the signal detection to form a new pulsesequence; the function of the new pulse sequence can be divided intothree parts: the first part is to obtain the singlets of the three ¹H onthe benzene ring of dopamine; the second part is to keep the singletstates of three ¹H on the benzene ring of the dopamine and filter theother non-single state signals because the nuclear spin singlet state isnot affected by the gradient field pulses and the CW decoupling pulse;the third part is to detect the singlet states of three ¹H on thebenzene ring; in the end, only the three ¹H signals on the benzene ringare kept, achieving the purpose of selective signal filtering; in thisprocess, it is necessary to continuously optimize the time of the CWpulse to achieve the best filtering efficiency.
 7. The method of claim6, as for the ¹H spectrum of dopamine described in step 1, the threesignals on the left side represent the three ¹H signals on the benzenering, the single peak in the middle is the water signal, and the ethylsignal of dopamine is on the right side; the J coupling values andchemical shifts between the three ¹H atoms on the benzene ring ofdopamine are obtained from the ¹H spectrum of dopamine.
 8. The method ofclaim 6, wherein step 2 is as follows: firstly, a 90-degree hard pulseis applied along x direction, after a delay time of τ₁, a 180-degreehard pulse is applied along the x direction; then, a delay time of(τ₁+τ₂) is applied, followed by a 90-degree hard pulse along the ydirection, and then a delay time of τ₂/2 namely τ₃ is applied; thefunction of this pulse is to prepare the singlet states of three ¹H onthe benzene ring of dopamine, which is called preparation pulse; becausethe singlet states cannot be directly detected, another pulse is neededto detect the singlets of the three ¹H of three-spin system on thebenzene ring of dopamine, which is called detection pulse; the form ofdetection pulse is as follows, after a delay time of τ₄, followed by a90-degree hard pulse along the y direction and then a delay time of τ₅is applied; next, take ADC sample immediately until the sampling signaldecay is completed; in this process, the values of τ₁ and τ₂ impact theefficiency of the singlet states consisting of three ¹H atoms on thebenzene ring of dopamine; in order to maximize the efficiency of thesinglet states, MATLAB software is used to calculate the values of τ₁and τ₂; first of all, 64 basic operators of the three-spin system areconstructed in the MATLAB script, and then the Hamiltonian of thethree-spin system consisting of three ¹H atoms on the benzene ring ofdopamine is written; finally, the operating operators corresponding tothe 90-degree hard pulse and the 180-degree hard pulse are obtained;then, the system is continuously evolving from the thermal equilibriumsignals under the operating operator and Hamiltonian of the hard pulse,and the evolution time τ₁ and τ₂ are continuously optimized to maximizethe singlet states preparation efficiency; similarly, on the basis ofgenerating singlet states, evolution time τ₄ and τ₅ are optimized tomaximize the singlet states detection efficiency; finally, the completepulse to prepare and detect three-spin system singlet states of dopamineis obtained by combining the pulse form of the two-spin system singletof the weakly coupled system and the calculated pulse parameters.
 9. Themethod of claim 6, as described in step 3, the singlet state of thethree-spin system consisting of three ¹H atoms on the benzene ring ofdopamine is prepared and detected; specifically, firstly, the completepulse obtained in step 2 is written into the computer by the NMRinstrument language; secondly, a D₂O aqueous solution of dopamine is putinto the magnetic resonance instrument, and then the field-locking,field-shimming, matching, and tuning are performed; finally, the radiofrequency center of the transmitter is set to the three ¹H on thebenzene ring of dopamine, and the complete pulse written into thecomputer is applied to prepare and detect the singlet states of thedopamine.
 10. The method of claim 6, as described in step 4, the CWdecoupling pulse and two gradient field pulses with different amplitudesare applied between the pulses for the singlet preparation and thepulses for the signal detection; this forms a new pulse module;specifically, the duration of the CW pulse is between 50 ms and 1 s; theamplitude varies from 1 watt to 15 watts; the duration of each of thetwo gradient field pulses with different amplitudes along the zdirection is between 1 ms and 5 ms, with the amplitude varying from 5 to10 Gauss/cm; this new pulse module including the CW decoupling pulse andthe two gradient field pulses with different amplitudes is written intothe computer by the NMR instrument language; then, the routineprocedures such as field-locking, field-shimming, matching, and tuningare performed; finally, the radio frequency center of the transmitter isset to the three ¹H on the dopamine benzene ring, and the new pulsesequence which has been written into the computer including the CWdecoupling pulse and the two gradient field pulses with differentamplitudes is applied to prepare and detect the singlet states ofdopamine.
 11. The method of claim 1, wherein the three ¹H spin couplingsystem on the benzene ring of dopamine is prepared into the nuclear spinsinglets in step C; there are several multiple spin systems in adopamine molecule; the spin system formed by the three ¹H spins on thebenzene ring of dopamine can be effectively prepared for the nuclearspin singlets.
 12. The method of claim 5, wherein the methodspecifically comprises the following steps: steps 1: put the D₂O aqueoussolution with a mass fraction of 2%-5% dopamine in the magneticresonance instrument, and apply a 90-degree hard pulse to the D₂Oaqueous solution of dopamine, which makes the ¹H signals excited, thenobtain the ¹H spectrum of dopamine, and in turn the chemical shifts ofthree ¹H on the benzene ring of dopamine and the J coupling values amongthe protons; steps 2: according to the pulse form of preparing thesinglets from the two-spin system of the weakly coupled system, for thethree-spin system consisting of three ¹H atoms on the benzene ring ofdopamine, the pulse parameters for the preparation and detection ofdopamine singlet in the three-spin system are calculated based onchemical shifts and J coupling values obtained from step 1 by usingMATLAB; as a result, the pulse sequence to prepare the nuclear spinsinglet states of three ¹H atoms on the dopamine benzene ring with themaximum efficiency can be obtained; steps 3: the complete pulse sequenceis obtained by combining the pulse form to prepare the singlet of thetwo-spin system of the weakly coupled system and the pulse parametersrequired for the preparation and detection of dopamine singlet in thethree-spin system calculated in step 2, which is applied to the dopamineD₂O aqueous solution to prepare and detect the singlet of the three-spinsystem consisting of three ¹H on the benzene ring; steps 4: on the basisof preparation and detection of dopamine singlet in step 3, two gradientfield pulses with different amplitudes and a continuous wave (CW)decoupling pulse are applied between the pulses for the singletpreparation and the pulses for the signal detection to form a new pulsesequence; the function of the new pulse sequence can be divided intothree parts: the first part is to obtain the singlets of the three ¹H onthe benzene ring of dopamine; the second part is to keep the singletstates of three ¹H on the benzene ring of the dopamine and filter theother non-single state signals because the nuclear spin singlet state isnot affected by the gradient field pulses and the CW decoupling pulse;the third part is to detect the singlet states of three ¹H on thebenzene ring; in the end, only the three ¹H signals on the benzene ringare kept, achieving the purpose of selective signal filtering; in thisprocess, it is necessary to continuously optimize the time of the CWpulse to achieve the best filtering efficiency.